Hydrolysis Step Research Articles

Addition of microbial consortium to the rice straw biomethanization: effect on specific methanogenic activity, kinetic and bacterial community

The biomethanization of lignocellulosic wastes remains an inefficient and complex process due to lignin structures that hinder the hydrolysis step, therefore, some treatments are required. This work describes the addition of an enriched microbial consortium in the biomethanization of rice straw. The experiment was carried out in lab batch reactors following two strategies: (i) pretreatment of rice straw for 48 h using the enriched microbial consortium (dilution 1:100), and (ii) addition of this enriched microbial consortium (dilution 1:100) directly to the anaerobic reactors (bioaugmentation). The kinetic behavior was described using three models. As a result, the microbial consortium molecular characterization showed 58 different bacterial species, predominantly the Lactobacillaceae family (45.7%), and the Clostridiaceae family (19.1%), which were responsible for the positive effect obtained by bioaugmentation with methane yield increases of 16% (290 LNCH4/kgVS) respect to the control. All kinetic models applied fitted the experimental data for cumulative methane production, although the modified Hill model showed the best fit. Bioaugmentation strategies demonstrate their effectiveness in lignocellulosic biodegradation, but the novelty of this research lies in the application of an enriched microbial consortium obtained by the authors through soil isolation techniques, which are very inexpensive and affordable for developing countries.

Towards a Quantitative Description of Proteolysis: Contribution of Demasking and Hydrolysis Steps to Proteolysis Kinetics of Milk Proteins.

The hydrolysis of proteins by proteases (proteolysis) plays a significant role in biology and food science. Despite the importance of proteolysis, a universal quantitative model of this phenomenon has not yet been created. This review considers approaches to modeling proteolysis in a batch reactor that take into account differences in the hydrolysis of the individual peptide bonds, as well as the limited accessibility (masking) for the enzymes of some hydrolysis sites in the protein substrate. Kinetic studies of the proteolysis of β-casein and β-lactoglobulin by various proteolytic enzymes throughout the whole degree of hydrolysis are reviewed. The two-step proteolysis model is regarded, which includes demasking of peptide bonds as a result of opening of the protein structure at the first stage, then hydrolysis of the demasked peptide bonds. To determine the kinetics of demasking, the shift in Trp fluorescence during opening of the protein substrate is analyzed. Two stages of demasking and secondary masking are also considered, explaining the appearance of unhydrolyzed peptide bonds at the end of proteolysis with decreasing enzyme concentrations. Proteolysis of a nanosized substrate is considered for the example of tryptic hydrolysis of β-CN micelles, leading to the formation and degradation of new nanoparticles and non-monotonic changes in the secondary protein structures during proteolysis.

Rapid kinetics of H+ transport by membrane pyrophosphatase: Evidence for a "direct-coupling" mechanism.

Stress resistance-conferring membrane pyrophosphatase (mPPase) found in microbes and plants couples pyrophosphate hydrolysis with H+ transport out of the cytoplasm. There are two opposing views on the energy-coupling mechanism in this transporter: the pumping is associated with either pyrophosphate binding to mPPase or the hydrolysis step. We used our recently developed stopped-flow pyranine assay to measure H+ transport into mPPase-containing inverted membrane vesicles on the timescale of a single turnover. The vesicles were prepared from Escherichia coli overproducing the H+-translocating mPPase of Desulfitobacterium hafniense. Pyrophosphate induced linear accumulation of H+ in the vesicles, without evident lag or burst. In contrast, the binding of three nonhydrolyzable pyrophosphate analogs essentially induced no H+ accumulation. These findings are inconsistent with the "pumping-before-hydrolysis" model of mPPase functioning and support the alternative model positing the hydrolysis reaction as the source of the transported H+ ions. mPPase is thus a first "directly-coupled" proton pump.

Impact of processing technologies on insect meal digestibility in rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax)

Abstract The objective of this study was to compare the apparent digestibility coefficients (ADCs) of nutrients and energy of three differently processed black soldier fly (BSF) meals in rainbow trout and European sea bass. The processing techniques included defatting with heat treatment followed by tricanter centrifugation, with or without an enzyme hydrolysis step, as well as microwave drying. In the experimental design, each processed BSF meal was mixed with a reference diet at a 30% inclusion level, using celite as an inert digestibility marker and fed to triplicate groups of rainbow trout and European sea bass. For the European sea bass trial, an additional diet with full fat oven-dried BSF meal was also evaluated. In rainbow trout, all the processed insect meals exhibited high digestibility, with no significant differences among the BSF meals. All the amino acids were highly digestible, with ADCs ranging from 84.8% to 93.6% for the essential amino acids and from 78% to 93.2% for the non-essential ones without significant differences between treatments (). In European sea bass, while fat and energy digestibility was similar, protein digestibility was significantly higher in all the processed BSF meals when compared to the oven-dried meal (). Between the two full fat insect meals, microwave drying significantly improved dry matter and protein digestibility of BSF meal when compared to oven-drying ( and , respectively). No differences were observed in the digestibility of the two defatted insect meals (), thus suggesting that enzymatic hydrolysis did not affect their digestibility in either fish species. This study suggests that processing techniques such as defatting and microwave drying can enhance the nutritional quality and digestibility of BSF meals and offers insights for optimizing insect-based feeds in aquaculture.

A Chemically Powered Rotary Molecular Motor Based on Reversible Oxazepine Formation

AbstractWhile biological machines are powered mainly by chemical transformations, chemically driven artificial rotary motor systems are very limited. Here, we report an aniline‐phenol‐based rotary molecular motor that operates via an information ratchet mechanism. The 360° directional rotation about a single covalent bond can be chemically driven by reversible oxazepine formation. Both the oxazepine formation and hydrolysis steps are kinetically gated via dynamic kinetic resolution, arising from the kinetic bias of chiral catalysts for enantiomers. Given the 95 % ee (97.5 : 2.5) and 88 % ee (94 : 6) of the individual gating steps of motor analogues, the overall directionality ratio could be calculated to be 91.7 : 8.3 (97.5 %×94 %≈91.7 %), which means that the motor will make one mistake (backward rotation) approximately every 11 to 12 turns.

A Chemically Powered Rotary Molecular Motor Based on Reversible Oxazepine Formation.

While biological machines are powered mainly by chemical transformations, chemically driven artificial rotary motor systems are very limited. Here, we report an aniline-phenol-based rotary molecular motor that operates via an information ratchet mechanism. The 360° directional rotation about a single covalent bond can be chemically driven by reversible oxazepine formation. Both the oxazepine formation and hydrolysis steps are kinetically gated via dynamic kinetic resolution, arising from the kinetic bias of chiral catalysts for enantiomers. Given the 95 % ee (97.5 : 2.5) and 88 % ee (94 : 6) of the individual gating steps of motor analogues, the overall directionality ratio could be calculated to be 91.7 : 8.3 (97.5 %×94 %≈91.7 %), which means that the motor will make one mistake (backward rotation) approximately every 11 to 12 turns.

Determination of trade-offs between 2G bioethanol production yields and pretreatment costs for industrially steam exploded woody biomass

Lignocellulosic biomass, including wood, can be transformed into bioethanol using steam explosion as pretreatment to improve saccharification and fermentation steps. Pretreatment is however the most expensive part of the process in terms of CAPEX and OPEX and requires to be optimized. In order to evaluate the link between pretreatment efficiency and cost, three contrasted wood species (oak, poplar and spruce) were pretreated with continuous steam explosion at pilot-scale following full factorial designs. Response surfaces obtained were combined with an economic assessment to determine trade-offs aiming at maximizing both fermentable sugars released during the enzymatic hydrolysis step and bioethanol yield during the fermentation step as well as minimizing costs of pretreatment in an industrial context. Results showed that bioethanol yields were highly dependent on wood species and that high severities of pretreatment were not the most relevant to apply. Optimal conditions of pretreatment corresponding to 70 % and 48 % of bioethanol producible from oak and poplar, respectively, were defined. The desirability function that has been modelled thus helps designing adapted pretreatment conditions regarding bioethanol production and process cost.

Multiomics-guided mining and characterization of epoxide hydrolase involved in camptothecin biosynthesis from Camptotheca acuminata

The 2,7-epoxy hydrolysis step is critical and inevitable for the biosynthesis of camptothecin (CPT). CPT-type drugs have excellent cytotoxic and antitumor activities. However, the genes responsible for this hydrolysis step remain unclear in Camptotheca acuminata Decne. In this study, multiomics resources of C. acuminata Decne have been utilized to mine and screen the genes involved in the epoxide hydrolase step. Three genes (CaEH1–CaEH3) have been identified, and their recombinant CaEH proteins have been prepared in a soluble form. All CaEHs display (S)-styrene oxide, (R)-styrene oxide, and trans-stilbene oxide oxirane ring-opening activities. Notably, CaEH1 displays excellent catalytic performance for (S)- and (R)-styrene oxides but poor enantioselectivity. On the other hand, CaEH2 and CaEH3 display a higher S isomer preference for styrene oxide. Furthermore, CaEH1–CaEH3 display strictosamide epoxide 2,7-epoxy ring opening activity. They exhibit inferior catalytic performance toward strictosamide epoxide compared to “slim” substrates but better catalytic performance for the larger substrates than characterized plant EHs. Functional verification in planta suggests that the newly identified CaEH1–CaEH3 are jointly responsible for CPT biosynthesis. These CaEH genes are expressed in all plantlet tissues and are enriched in the leaves. Evolutionary analysis indicates that CaEH1–CaEH3 originate from different ancestral EH genes. The convergent evolution of these CaEH genes likely results in the homofunctionalization of CaEH1–CaEH3. Overall, this study reveals one of the previously unexplored biosynthetic steps of camptothecin in C. acuminata.

Quantifying the reversibility of ATP hydrolysis in rabbit skeletal myosin subfragment 1 using a luciferase assay

BackgroundIt is known that the adenosine triphosphate (ATP) hydrolysis in a number of ATPases is reversible. Radioactive methods are standard in measuring ATPase reversibility. We used myosin as a well-studied model enzyme to develop a luciferase-based assay to quantify the reversibility of ATP hydrolysis. In myosin, ATP bound to the active site is hydrolyzed into adenosine diphosphate (ADP) and inorganic phosphate (Pi) and recombined back into ATP multiple times before products of hydrolysis are released. The reversibility of ATP hydrolysis by skeletal myosin was previously confirmed using radioactively labeled isotopes. Transient kinetics studies indicated that the ATP hydrolysis step is temperature-sensitive, with a dissociation equilibrium constant of 1.6 at 3 °C. Consequently, the association equilibrium constant at this temperature is 0.63. The goal of our work was two-fold, (a) to develop a luciferase-based assay to measure the equilibrium constant of enzymatic ATP hydrolysis, eliminating the need for radioactively labeled Pi, and (b) refine the value of the association equilibrium constant of the myosin ATPase hydrolysis step.MethodsIn this assay, a reaction mixture containing myosin and saturating levels of ADP and Pi was incubated to reach equilibrium, then the reaction was terminated, and the amount of ATP produced by myosin was quantified using the luciferase assay. The equilibrium constant of ATP hydrolysis was defined as the ratio of ATP to ADP bound to myosin.ResultsWe obtained a value of 0.78 ± 0.14 for the association equilibrium constant of ATP hydrolysis at 0 °C. 50 μM ADP bound to myosin is turned into 21.9 ± 3.0 μM ATP. Our result is in excellent agreement with the literature data, supporting the viability of the new methodology.DiscussionThis methodology allows a more accessible and safe option to measure ATP production and reversibility of ATPase enzymes. Standard laboratory equipment is utilized in the assay, and the number of steps in the developed assay is reduced significantly compared to the radioactive method.

Enhanced anaerobic digestion of freezing and thawing pretreated cow manure with increasing solid content: kinetics and microbial community dynamics

High solid anaerobic digestion has proved the mainstream technology for the treatment of organic wastes. However, the high molecular weight and complex lignocellulosic structure of cow manure (CM) make it indigestible and inefficient, leading to limit the hydrolysis step of anaerobic digestion at high solid content. To mitigate this bottleneck, an improved cost-effective freezing and thawing pretreatment technique was proposed in this study. The freezing and thawing pretreatment of raw CM without any dilution was done for 20 days. The maximum cumulative methane yield (487 mL CH4 g− 1VS) was achieved at a total solid (TS) of 5% followed by TS of 10% and 15%, which was 13%, 20% and 21% higher than obtained from untreated CM, respectively. The kinetic results revealed that the biodegradable materials could be utilized at increasing TS with decreasing hydrolysis rate. The pretreatment significantly enhanced the methylotrophic methanogenic pathway during high solid anaerobic digestion, which was contrary to the general concept that the process is usually dominated by acetoclastic and hydrogenotrophic methanogens. This study is very important to understand the effect of solid content but also important to understand the effect of freezing and thawing pretreatment on process parameters and microbial community dynamics in high solid anaerobic digestion.

Regarding Bioanalysis Lasting a Few Minutes: Automated Cooling-SPME and Fast-GC for Urinary 2-Phenyl-2-Propanol Monitoring.

An innovative SPME head space GC-MS method, in cooling mode, using a fully automated routine, was developed to detect 2-phenyl-2-propanol, a representative urinary metabolite of cumene. Following an acid hydrolysis and derivatization step with lowered quantities of reagents, acetic anhydride and pyridine, a 30 μm polydimethylsiloxane SPME fiber was used to sample derivatized 2-phenyl-2-propanol, such as benzenemethanol,α,α-dimethyl-acetate, from the headspace. Performances of the method, optimized through experimental design, provide an LOD of 0.034 mg/L and an LOQ 0.10 mg/L, with a short sampling time necessary per sample. The method, developed on standard solutions, will be applied to both occupationally exposed and non-exposed populations.

Remote Sulfonylation of Anilines with Sodium Sulfifinates Using Biomass-Derived Copper Catalyst

A biomass-based catalyst, CuxOy@CS-400, was employed as an excellent recyclable heterogeneous catalyst to realize the sulfonylation reaction of aniline derivatives with sodium sulfinates. Various substrates were compatible, giving the desired products moderate to good yields at room temperature. In addition, this heterogeneous copper catalyst was also easy to recover and was recyclable up to five times without considerably deteriorating in catalytic efficiency. Importantly, these sulfonylation products were readily converted to the corresponding 4-sulfonyl anilines via a hydrolysis step. The method offers a unique strategy for synthesizing arylsulfones and has the potential to create new possibilities for developing heterogeneous copper-catalyzed C-H functionalizations.

Endosomal actin branching, fission, and receptor recycling require FCHSD2 recruitment by MICAL-L1.

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process.

1O2 and Base Assisted Oxidative Conversion of β-Enaminoesters to α-Acyloxy-β-ketoesters under Visible Light Irradiation.

Singlet oxygen (1O2) and base assisted conversion of β-enaminoesters to α-acyloxy-β-ketoesters is demonstrated under visible light irradiation. The reaction involves formation of an imine intermediate via ene-type pathway initiated by 1O2 followed by base promoted dimerization and hydrolysis steps. The method is mild, environmentally friendly, requires air as the oxidant, and gives the products in moderate to high yields.

A multistage biogas slurry reflux and spray anaerobic digestion reactor for high solid anaerobic digestion: Performance and application evaluation

In this study, a new vertical plug-flow continuous dry anaerobic digestion reactor equipped with a spray percolation system was designed for rice straw and cow manure in dry AD. The findings indicated that the methane yield was 290.63 ± 19.16 mL/g VS when the optimal spray time (9 times/d) was used in batch AD. In addition, the results of continuous experiments under optimal spray time conditions showed that the volumetric methane production rate of the system was 1.55 L/(L·d) when the organic loading rate was 20 g VS/(L·d). In addition, hydrolysis and acidification steps were enhanced. This study demonstrated the potential of the new reactor for stable operation of high OLR dry AD. Digestate is already used as a substitute for synthetic fertilizers precisely because of the environmental benefits associated with its agronomic use.

Rational Engineering of a β-Glucosidase (H0HC94) from Glycosyl Hydrolase Family I (GH1) to Improve Catalytic Performance on Cellobiose.

The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate p-nitrophenyl β-d-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. We report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 μmol/min/mg), a 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in catalytic efficiency (236.65 mM-1 s-1) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase β-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.

Green directional conversion of food waste into glycerol in a two-step differentiation enzymolysis and facultative fermentation using Saccharomyces cerevisiae: Performance assessment and mechanism

Valorization of food waste into high value-added glycerol was designed and prepared via directional green transformation using multi-enzymolysis saccharification coupled facultative fermentation by Saccharomyces cerevisiae. The optimal conditions for the first step of multienzyme hydrolysis and the second step of facultative fermentation were 55 °C and pH 5.0 for 6 h, 30 °C and pH 7.0 for 70 h, respectively. The glycerol yield efficiency and kinetic rate constant were 73.6 % (w/w) and 0.681 h−1 for dry component of food waste, while the other 27.4 % was utilized as the growth energy substrate. The key points of transformation were the synergistic directed enzymolysis to produce 3 % glycerol and 95 % reducing sugars. Furthermore, the reducing sugars were converted into glycerol by switch the metabolic pathway from ethanol into glycerol through the quantitative expression of functional gpd1 and gpp2 genes activated GPD and GPP enzymatic activity and synchronous inhibition of adh gene for glycerol production with Na2SO3 regulation. The economic income was 135.52 USD/ton for converting 80 % water content of kitchen waste into glycerol. This finding provided an economic value-added approach to produce high-value glycerol for cost-effective recycling of food waste using commercial yeast.

Evaluation of a Benzodiazepine Immunoassay for Urine Drug Testing in Clinical Specimens.

Benzodiazepines are commonly prescribed medications frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated benzodiazepine metabolites, leading to false negatives. Clinical laboratories have addressed this deficiency by creating laboratory-developed tests (LDTs) that incorporate a beta-glucuronidase hydrolysis step to increase the clinical sensitivity of these assays. Performance characteristics of 2 FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a previously described benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 clinical urine specimens. The positive immunoassay cutoff was set at 200 ng/mL of nordiazepam and results were compared to an LC-MS/MS benzodiazepine LDT. Clinical sensitivity, specificity, precision, and immunoassay cross-reactivity were determined for all 3 immunoassays. The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all 3 assays. Immunoassay response of the Benzodiazepines II assay was greater across the range of concentrations tested (100-1000 ng/mL) relative to the other immunoassays and was the most sensitive immunoassay for the detection of lorazepam glucuronide. The Benzodiazepines II immunoassay demonstrated the greatest clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The incorporation of beta-glucuronidase was crucial, as the Benzodiazepines II and LDT immunoassays demonstrated superior clinical sensitivity when compared to the Benzodiazepines Plus immunoassay that does not incorporate a beta-glucuronidase hydrolysis step.

One‐Pot Cascade Reaction from Epoxide to Carboxylic Acids Using Bifunctional Fe‐ZSM‐5

AbstractThe quest for sustainable synthesis of carboxylic acids has led to the exploration of innovative routes and catalysts for the oxidative cleavage of carbon−carbon double bonds, prevalent in bio‐derived molecules. This process involves multiple steps including epoxidation, hydrolysis and vicinal diol cleavage, often catalysed by unsustainable homogeneous or noble‐metal catalysts. In this study, we present the utilization of Fe‐ZSM‐5 as a versatile bifunctional catalyst capable of catalysing both hydrolysis and vicinal diol cleavage steps, utilizing propylene oxide as model compound and H2O2 as oxidizing agent. The prepared Fe‐ZSM‐5 features the zeolite intrinsic Lewis and Brønsted acidity functions, essential for the hydrolysis step, as well as high fractions of monomeric FeOx species, crucial for the subsequent vicinal diol cleavage, leading to streamlined carboxylic acids formation in a one‐pot, two‐step process. Moreover, by combining Fe‐ZSM‐5 with TS‐1, renowned for its epoxidation activity, we demonstrate the complete cascade reaction in one pot, under mild conditions, starting from ethylene. Our study expands the utility of cost‐effective zeolite‐based catalysts enriched with abundant Fe metal for the one‐pot oxidative cleavage of C=C double bonds, offering a promising pathway towards sustainable carboxylic acid synthesis.

Enhanced cellobiose hydrolysis over fluorine-modulated carbon-based solid acid catalysts

In this study, we synthesized novel carbon-based solid acid catalysts using plasma engineering by integrating sulfonic acid groups as primary catalytic sites and supplemented these with fluorine-containing, chlorine-containing, and other oxygenated functional groups (OFGs). Notably, the catalyst with the fluorine-containing functional groups results superior glucose yield (58.2 %) than that containing only OFGs (37.6 %). Theoretical analysis of the reaction energetics revealed that the cleavage of cellobiose's 1,4-O linkage to form two glucose molecules potentially constitutes the rate-determining step (RDS) in cellobiose hydrolysis. The reaction energy for this RDS was found to increase in the sequence of G–SO3H–F, G–SO3H–Cl, and G-SO3H, consistent with the experimentally obtained catalytic activity trends. Through extensive characterization, including textural analysis, surface chemistry, and density functional theory calculations, we identified that the –F groups are the key to strengthening cellulose–catalyst interactions. This study is the first study to demonstrate the potential of fluorine-modulated carbon-based solid acid catalysts for upgrading cellulosic biomass to value-added compounds.